Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved proteins necessary for intracellular vesicular trafficking and fusion throughout the secretory and endocytic pathways. SNAREs have been extensively characterized in synaptic vesicles, where they are required for synaptic vesicle fusion and neurotransmitter release. Two plasma membrane proteins, SNAP-25 and syntaxin- 1, and the synaptic vesicle protein synaptobrevin-2 (Syb2/VAMP2) mediate this process (Jahn and Scheller, 2006). Furthermore, a specific role in synaptic vesicle exo-endocytosis coupling has been ascribed to Syb2, but not SNAP-25. Both Syb2 and SNAP-25 are required for normal spontaneous and evoked neurotransmission, but each are relatively more important for stimulus-evoked transmission (Schoch et al., 2001; Bronk et al., 2007). Therefore, physiological transmission must partially depend on additional unknown SNARE proteins since some transmission remains in synapses from animals lacking Syb2 or SNAP-25, and these alternative SNAREs may preferentially support spontaneous transmission. Syb2 is a member of a family of structurally related SNARE proteins, many of which are also present in synaptic vesicles, albeit at reduced levels (Takamori et al., 2006). A subgroup of this family, including Vti1a and VAMP7, possesses unique N- terminal extensions that may regulate these proteins' ability to support neurotransmission (Wang and Tang, 2006; Rossi et al., 2004). Based on their domain homologies to the canonical exocytic SNAREs and their localization in presynaptic vesicles, VAMP7 and Vti1a are likely candidates to support transmission in the absence of Syb2 or SNAP-25. The roles of Vti1a and VAMP7 in central synaptic transmission will be studied using both gain- and loss-of-function approaches in combination with state of the art live-cell imaging technologies and electrophysiology. Fusion proteins of VAMP7 and Vti1a coupled to pH-sensitive GFP will be used to measure these proteins' stimulation-dependent or -independent trafficking and ability to support exo- endocytosis coupling in synaptic vesicles (Aim 1). Whole-cell patch-clamp recordings will directly demonstrate functions of these proteins in synaptic transmission (Aim 2). Key series of experiments will assess the ability of VAMP7 or Vti1a to functionally substitute for Syb2 or SNAP-25 as well as the regulatory roles of their N-termini in this process. Preliminary imaging data show specific trafficking of Vti1a during spontaneous activity, putatively identifying this protein as a unique SNARE capable of supporting a specific form of neurotransmission. Importantly, alterations in synaptic vesicle trafficking may contribute to the pathological effects of some neurodegenerative or psychiatric diseases by modifying presynaptic or postsynaptic plasticity. A thorough understanding of this basic process will complement ongoing research in these areas and may elucidate novel therapeutic targets. PUBLIC HEALTH RELEVANCE: Synaptic transmission is a fundamental property of neurons, and alterations in this process are associated with a number of pathological neurodegenerative and psychiatric disorders, including but not limited to lysosomal storage disorders, Parkinson's disease, bipolar disorder, and schizophrenia. The proposed experiments will further elucidate molecular mechanisms underlying presynaptic vesicle trafficking and fusion under normal conditions and promote the discovery of novel therapeutic targets for a variety of human neurological diseases.